The goal of the proposed research is to develop 3'-phosphono- oligodeoxynucleotides as a new class of oligodeoxynucleotide analogs that have been designed to have superior performance as antisense oligonucleotide inhibitors of HIV replication. In these analogs, the 3' oxygen of the phosphodiester linkage has been replaced by a methylene (CH2) group, which is expected to provide resistance to the action of nucleases without altering the structural properties of he duplex. Before these analogs can be examined for their potential in antisense DNA therapy, however, a practical means for their synthesis must be developed. Three synthetic approaches are proposed for the preparation of monomers that may be used for the solid-phase synthesis of oligomers possessing any number of phosphonate internucleotide linkages. Although coupling methodologies analogous to the phosphotriester approach will be initially employed to prepare oligomers for biochemical evaluation, the synthesis of phosphinate monomers, which are directly analogous to the H-phosphonate monomers currently employed for the automated synthesis of oligonucleotides, will also be explored. A practical synthesis of the phosphinate monomers will allow direct and convenient access to the 3'-phosphono-oligodeoxynucleotide analogs. The synthetic oligonucleotide analogs will be subjected to a critical evaluation of their competency to serve as oligodeoxynucleotide surrogates. Thus, duplexes of the analog oligomers with complementary oligodeoxynucleotides and oligoribonucleotides will be examined for their structural correspondence with normal duplexes. The experimental techniques employed for this comparison will be measurement of Tm's and two-dimensional nuclear magnetic resonance. The biological competency of the analogs will also be addressed. The analog's resistance to nucleases will be experimentally quantified, and the ability for the oligonucleotide analogs to serve as substrates for DNA processing enzymes such as T4 polynucleotide kinase, T4 ligase, and polynucleotide terminal transferase will be addressed. In addition, the competency of the analogs to serve as templates for DNA polymerase and RNA polymerase will be determined, as will the activity of the RNase H activity of reverse transcriptase towards analog oligomer-RNA duplexes. Together, these physical and biochemical studies will define the degree to which the phosphonate analogs mimic normal DNA. Concurrent with the characterization of the oligonucleotide analogs, oligomers will be prepared with sequences complementary to target regions of HIV RNA. These antisense oligomers will be tested in cell culture for anti-HIV activity by Dr. P. S. Sarin at NIH.